Two new human DnaJ-like proteins

ABSTRACT

The invention provides a two new human DnaJ-like proteins (HSPJ1 or HSPJ2) and polynucleotides which identify and encode HSPJ1 or HSPJ2. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of HSPJ1 or HSPJ2.

This application is a divisional application of U.S. application Ser.No. 09/235,373, filed Jan. 20, 1999, which is a divisional of U.S.application Ser. No. 08/868,288, filed Jun. 3, 1997, issued Jul. 13,1999, as U.S. Pat. No. 5,922,567.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of twonew human DnaJ-like proteins and to the use of these sequences in thediagnosis, prevention, and treatment of cancer and inflammatory andimmune disorders.

BACKGROUND OF THE INVENTION

Induction of heat shock proteins (Hsps), a class of molecularchaperones, is a physiological and biochemical response to abruptincreases in temperature or exposure to a variety of other metabolicinsults including heavy metals, amino acid analogs, toxins, and tooxidative stress. This response occurs in all prokaryotic and eukaryoticcells and is characterized by repression of normal protein synthesis andinitiation of transcription of Hsp-encoding genes. Under normal ornonstressed conditions, constitutively expressed Hsps facilitate properprotein folding and maturation, promote protein translocation acrossmembranes, and regulate hormone receptor and protein kinase activity(Hightower, L. E., et al. (1991) Cell, 66: 191-197).

During cellular stress, Hsps form a complex with proteins that misfoldor unfold, either "rescuing" these proteins from irreversible damage orincreasing their susceptibility to proteolytic attack. Overexpression ofHsps in transgenic mice and rats, or prior heat treatment of normalanimals to induce Hsps, protects the heart muscle from ischemic injury.Both heat shock-induced and exogenous Hsps protect smooth muscle cellsfrom serum deprivation-induced cell death. Overexpression of Hsps alsoprotects murine fibroblasts from both UV light injury andproinflammatory cytokines released during UV exposure. Specific Hspsbind immunosuppressive drugs and may play a role in modulation of immuneresponses. Hsps expressed in cancer cells can protect the cancer cellsfrom the cytotoxic effects of drugs used in anticancer therapies. Hspsisolated from tumor cells, when purified and used as antigens, have beenshown to provide immunity to the tumors from which they are isolated(Udono, H., et al. (1994) J. Immunol. 152: 5398-5403; Young R. A. (1990)Annu. Rev. Immunol. 8: 401-420; Marber, M. S., et al. (1995) J. Clin.Invest. 95: 1446-1456; Simon, M. M., et al. (1995) J. Clin. Invest. 95:926-933).

Several of the constitutive Hsp genes are located in the majorhistocompatibility complex on chromosome 6, and members of the Hspfamily play roles in T-cell mediated regulation of inflammation andimmune recognition. Hsps bind to steroid hormone receptors, represstranscription in the absence of the ligand, and provide the properfolding of the ligand-binding domain in the presence of the hormone.Heat shock treatment of B-cells enhances processing of antigen and theassembly and function of MHC class II molecules (Sargent, C., A. et al(1989) Proc. Natl. Acad. Sci. 86: 1968-1972; Fang, Y., et al. (1996) J.Biol. Chem. 271: 28697-28702; Hendrick, J. P., et al (1993) Proc. Natl.Acad. Sci. 90: 10216-10220).

Knockout mice are providing additional information on the roles of Hsps.For example, female homozygous knockout mice for Hsp70 are found toundergo normal meiosis and are fertile. In contrast, the homozygous maleknockout mice lack postmeiotic spermatids and mature sperm and areinfertile (Dix, D. J. et al. (1996) Proc. Nat. Acad. Sci. 93:3264-3268).

Hsps function in a variety of necessary cellular processes includingprotein translocation across membranes of cell organelles, nascentprotein folding and multiunit protein assembly, antigen presentation,protein degradation in the lysosome, and uncoating of clathrin-coatedvesicles. They are located in all major cellular compartments andfunction as monomers, multimers, or in complexes with other cellularproteins, which may determine the rate and specificity of the Hspaction. The yeast and bacterial homologues of the human Hsp70 functionas a complex with the DnaJ gene product to accelerating the rate of ATPhydrolysis during protein folding and protein complex assembly. Humanhomologues of the DnaJ protein have recently been characterized and arefound be strongly induced by heat shock and to have sequence similaritywith the DnaJ protein family. The DnaJ homologue, hsp40, was shown tocolocalize with hsp70 in the nuclei and nucleoli of heat-shocked HeLacells. Two other homologues, HSJ1a and HSJ1b, are expressed primarily inthe human hippocampus and frontal cortex (Ohtsuka, K. (1993) Biocem.Biophys. Res. Com. 197: 235-240 and Cheetham, M., E., et al. (1992)Biochem. J. 284: 469-476).

The discovery of two new DnaJ-like proteins and the polynucleotidesencoding them satisfies a need in the art by providing new compositionswhich are useful in the diagnosis, prevention and treatment of cancer,inflammatory, and immune disorders.

SUMMARY OF THE INVENTION

The invention features two substantially purified polypeptides, newhuman DnaJ-like proteins (HSPJ1 and HSPJ2), having the amino acidsequence shown in SEQ ID NO:1, (HSPJ1), and SEQ ID NO:3, (HSPJ2), orfragments thereof.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:1, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:1, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO.2 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:2.

In another aspect the invention provides a composition comprising anisolated and purified polynucleotide sequence comprising the complementof SEQ ID NO:2, or fragments or variants thereof. The invention alsoprovides a polynucleotide sequence comprising the complement of SEQ IDNO:2.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding HSPJ1 under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified HSP1 having the amino acid sequence of SEQ IDNO:1 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist which decreases theactivity of a polypeptide of SEQ ID NO:1. In one aspect the inventionprovides a purified antibody which binds to a polypeptide comprising atleast a fragment of the amino acid sequence of SEQ ID NO:1.

Still further, the invention provides a purified agonist which modulatesthe activity of the polypeptide of SEQ ID NO:1.

The invention also provides a method for treating or preventing cancercomprising administering to a subject in need of such treatment aneffective amount of a pharmaceutical composition comprising anantagonist to HSPJ1.

The invention also provides a method for treating or preventinginflammatory and immune disorders comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising an antagonist to HSPJ1.

The invention also provides a method for treating or preventing tissuedamage comprising administering to a subject in need of such treatmentan effective amount of a pharmaceutical composition comprising purifiedHSPJ1.

The invention also provides a method for detecting a polynucleotidewhich encodes HSPJ1 in a biological sample comprising the steps of: a)hybridizing a polynucleotide sequence complementary to HSPJ1 (SEQ IDNO:1) to nucleic acid material of a biological sample, thereby forming ahybridization complex; and b) detecting the hybridization complex,wherein the presence of the complex correlates with the presence of apolynucleotide encoding HSPJ1 in the biological sample. In a preferredembodiment, prior to hybridization, the nucleic acid material of thebiological sample is amplified by the polymerase chain reaction.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:3 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:3, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:3, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO.4 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:4.

In another aspect the invention provides a composition comprising anisolated and purified polynucleotide sequence comprising the complementof SEQ ID NO:4, or fragments or variants thereof. The invention alsoprovides a polynucleotide sequence comprising the complement of SEQ IDNO:4.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:3 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding HSPJ2 under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified HSPJ2 having the amino acid sequence of SEQ IDNO:3 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist which decreases theactivity of a polypeptide of SEQ ID NO:3. In one aspect the inventionprovides a purified antibody which binds to a polypeptide comprising atleast a fragment of the amino acid sequence of SEQ ID NO:3.

Still further, the invention provides a purified agonist which modulatesthe activity of the polypeptide of SEQ ID NO:3.

The invention also provides a method for treating or preventing cancercomprising administering to a subject in need of such treatment aneffective amount of a pharmaceutical composition comprising anantagonist to HSPJ2.

The invention also provides a method for treating or preventinginflammatory and immune disorders comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising an antagonist to HSPJ2.

The invention also provides a method for treating or preventing tissuedamage comprising administering to a subject in need of such treatmentan effective amount of a pharmaceutical composition comprising purifiedHSPJ2.

The invention also provides a method for detecting a polynucleotidewhich encodes HSPJ2 in a biological sample comprising the steps of: a)hybridizing a polynucleotide sequence complementary to HSPJ2 (SEQ IDNO:3) to nucleic acid material of a biological sample, thereby forming ahybridization complex; and b) detecting the hybridization complex,wherein the presence of the complex correlates with the presence of apolynucleotide encoding HSPJ2 in the biological sample. In a preferredembodiment, prior to hybridization, the nucleic acid material of thebiological sample is amplified by the polymerase chain reaction.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of HSPJ1. The alignment was producedusing MACDNASIS PRO™ software (Hitachi Software Engineering Co. Ltd. SanBruno, Calif.).

FIGS. 2A and 2B show the amino acid sequence alignment between HSPJ1(SEQ ID NO:1) and DNAJ-2 (GI 306714; SEQ ID NO:5), produced using themultisequence alignment program of DNASTAR™ software (DNASTAR Inc,Madison, Wis.).

FIGS. 3A, 3B, 3C, and 3D show the amino acid sequence (SEQ ID NO:3) andnucleic acid sequence (SEQ ID NO:4) of HSPJ2. The alignment was producedusing MACDNASIS PRO™ software (Hitachi Software Engineering Co. Ltd. SanBruno, Calif.).

FIGS. 4A and 4B show the amino acid sequence alignments among HSPJ2 (SEQID NO:3), HSJ1a (GI 32469; SEQ ID NO:6) and HSJ1b (GI 32470; SEQ IDNO:7), produced using the multisequence alignment program of DNASTAR™software (DNASTAR Inc, Madison, Wis.).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

HSPJ, as used herein, refers to the amino acid sequences ofsubstantially purified HSPJ obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

The term "agonist", as used herein, refers to a molecule which, whenbound to HSPJ, increases or prolongs the duration of the effect of HSPJ.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of HSPJ.

An "allele" or "allelic sequence", as used herein, is an alternativeform of the gene encoding HSPJ. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

"Altered" nucleic acid sequences encoding HSPJ as used herein includethose with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent HSPJ. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding HSPJ,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding HSPJ. The encoded protein may also be "altered" and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent HSPJ.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of HSPJ is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

"Amino acid sequence" as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragment thereof, and to naturallyoccurring or synthetic molecules. Fragments of HSPJ are preferably about5 to about 15 amino acids in length and retain the biological activityor the immunological activity of HSPJ. Where "amino acid sequence" isrecited herein to refer to an amino acid sequence of a naturallyoccurring protein molecule, amino acid sequence, and like terms, are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

"Amplification", as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "antagonist", as used herein, refers to a molecule which, whenbound to HSPJ, decreases the amount or the duration of the effect of thebiological or immunological activity of HSPJ. Antagonists may includeproteins, nucleic acids, carbohydrates, or any other molecules whichdecrease the effect of HSPJ.

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind HSPJ polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal can be derived from thetranslation of RNA or synthesized chemically and can be conjugated to acarrier protein, if desired. Commonly used carriers that are chemicallycoupled to peptides include bovine serum albumin and thyroglobulin,keyhole limpet hemocyanin. The coupled peptide is then used to immunizethe animal (e.g., a mouse, a rat, or a rabbit).

The term "antigenic determinant", as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term "antisense", as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term "antisense strand" is used in reference toa nucleic acid strand that is complementary to the "sense" strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation "negative" is sometimes used in referenceto the antisense strand, and "positive" is sometimes used in referenceto the sense strand.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic HSPJ, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

A "composition comprising a given polynucleotide sequence", as usedherein, refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding HSPJ (SEQ ID NO:1 or SEQ ID NO:3) or fragments thereof (e.g.,SEQ ID NO:2 and fragments thereof) may be employed as hybridizationprobes. The probes may be stored in freeze-dried form and may beassociated with a stabilizing agent such as a carbohydrate. Inhybridizations, the probe may be deployed in an aqueous solutioncontaining salts (e.g., NaCl), detergents (e.g., SDS) and othercomponents (e.g., Denhardt's solution, dry milk, salmon sperm DNA,etc.).

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, has been extended usingXL-PCR™ (Perkin Elmer, Norwalk, CT) in the 5' and/or the 3' directionand resequenced, or has been assembled from the overlapping sequences ofmore than one Incyte Clone using a computer program for fragmentassembly (e.g., GELVIEW™ Fragment Assembly system, GCG, Madison, Wis.).Some sequences have been both extended and assembled to produce theconsensus sequence.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding HSPJ in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

A "deletion", as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to HSPJ or theencoded HSPJ. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term "substantiallyhomologous." The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Human artificial chromosomes (HACs) are linear microchromosomes whichmay contain DNA sequences of 10K to 10M in size and contain all of theelements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat Genet. 15:345-355).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

"Microarray" refers to a high-density array of distinct polynucleotidesor oligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

The term "modulate", as used herein, refers to a change in the activityof HSPJ. For example, modulation may cause an increase or a decrease inprotein activity, binding characteristics, or any other biological,functional or immunological properties of HSPJ.

"Nucleic acid sequence", as used herein, refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. "Fragments" are thosenucleic acid sequences which are greater than 60 nucleotides than inlength, and most preferably includes fragments that are at least 100nucleotides or at least 1000 nucleotides, and at least 10,000nucleotides in length.

The term "oligonucleotide" refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or hybridization assays. As used herein,oligonucleotide is substantially equivalent to the terms "amplimers","primers", "oligomers", and "probes", as commonly defined in the art.

"Peptide nucleic acid", PNA, as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast five nucleotides in length linked to a peptide backbone of aminoacid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from five amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length HSPJ1 and fragments thereof.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding HSPJ, orfragments thereof, or HSPJ itself may comprise a bodily fluid, extractfrom a cell, chromosome, organelle, or membrane isolated from a cell, acell, genomic DNA, RNA, or cDNA(in solution or bound to a solid support)a tissue, a tissue print, and the like.

The terms "specific binding" or "specifically binding", as used herein,refers to that interaction between a protein or peptide and an agonist,an antibody and an antagonist. The interaction is dependent upon thepresence of a particular structure (i.e., the antigenic determinant orepitope) of the protein recognized by the binding molecule. For example,if an antibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The terms "stringent conditions" or "stringency", as used herein, referto the conditions for hybridization as defined by the nucleic acid,salt, and temperature. These conditions are well known in the art andmay be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

A "variant" of HSPJ, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The Invention

The invention is based on the discovery of two new human DnaJ-likeproteins (hereinafter referred to as HSPJ1 and HSPJ2, and collectively,as HSPJ), the polynucleotides encoding HSPJ, and the use of thesecompositions for the diagnosis, prevention, or treatment of cancer andinflammatory and immune disorders.

Nucleic acids encoding the HSPJ1 of the present invention were firstidentified in Incyte Clone 136466 from the synovial membrane tissue cDNAlibrary (SYNORAB01) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 136466 (SYNORAB01), 1450458 (PENITUT01), 1468130 (PANCTUT02),403306 (TMLR3DT01), 568426 (MMLR3DT01), 696229 (SYNORAT03), and 1255031(LUNGFET03).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A through 1D.HSPJ1 is 358 amino acids in length and has a potential DnaJ domainbetween residues F₆₇ and Y₈₆. As shown in FIGS. 2A and 2B, HSPJ1 haschemical and structural homology with DNAJ-2 (GI 306714; SEQ ID NO:3).In particular, HSPJ1 and DNAJ-2 share 36% identity. Northern analysisshows the expression of this sequence in various libraries, at least 46%of which are immortalized or cancerous. Of particular note is theexpression of HSPJ1 in disorders which involve the inflammation andimmune response (18%).

Nucleic acids encoding the HSPJ2 of the present invention were firstidentified in Incyte Clone 260873 from the hNT2 cDNA library (HNT2RAT01)using a computer search for amino acid sequence alignments. A consensussequence, SEQ ID NO:4, was derived from the following overlapping and/orextended nucleic acid sequences: Incyte Clones 260873, 269831(HNT2RAT01), 264667 (HNT2AGT01), 448764 (TLYMNOT02), 1003613 10(BRSTNOT03), 1503542 (BRAITUT07), 112064 (PITUNOT01), and 1440865(THYRNOT03).

In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:3, as shown in FIGS. 3Athrough 3D. HSPJ2 is 330 amino acids in length and has a potential DnaJdomain between residues F₄₆ and Y₆₅. As shown in FIGS. 4A and 4B, HSPJ2has chemical and structural homology with HSJ1a (GI 32469; SEQ ID NO:6)and HSJ1b (GI 32470; SEQ IN NO:7). In particular, HSPJ2 and HSJ1a share46% identity, while HSPJ2 and HSJ1b share 44% identity. Northernanalysis shows the expression of this sequence in various libraries, atleast 59% of which are immortalized or cancerous.

The invention also encompasses HSPJ variants. A preferred HSPJ variantis one having at least 80%, and more preferably 90%, amino acid sequenceidentity to the HSPJ1 or HSPJ2 amino acid sequence (SEQ ID NO:1, SEQ IDNO:3). A most preferred HSPJ variant is one having at least 95% aminoacid sequence identity to SEQ ID NO:1 or SEQ ID NO:3.

The invention also encompasses polynucleotides which encode HSPJ.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of HSPJ can be used to produce recombinant molecules whichexpress HSPJ. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A through 1D and the polynucleotide comprising thenucleic acid sequence of SEQ ID NO:4 as shown in FIG. 3A through 3D.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding HSPJ, some bearing minimal homology to the nucleotide sequencesof any known and naturally occurring gene, may be produced. Thus, theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence ofnaturally occurring HSPJ, and all such variations are to be consideredas being specifically disclosed.

Although nucleotide sequences which encode HSPJ and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HSPJ under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HSPJ or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HSPJ and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode HSPJ and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding HSPJ or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency as taught in Wahl, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification Systemmarketed by Gibco/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

The nucleic acid sequences encoding HSPJ may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 Primer Analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1: 111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5' non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER and SEQUENCE NAVIGATOR,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HSPJ may be used in recombinant DNAmolecules to direct expression of HSPJ, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressHSPJ.

As will be understood by those of skill in the art, it may beadvantageous to produce HSPJ-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter HSPJ encodingsequences for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HSPJ may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HSPJ activity, it may be useful toencode a chimeric HSPJ protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HSPJ encoding sequence and theheterologous protein sequence, so that HSPJ may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding HSPJ may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of HSPJ, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of HSPJ, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active HSPJ, the nucleotide sequencesencoding HSPJ or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HSPJ andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding HSPJ. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The invention is not limited by the host cell employed.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are Ipreferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding HSPJ,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for HSPJ. For example, when largequantities of HSPJ are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding HSPJ may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding HSPJ may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-31 1). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196.

An insect system may also be used to express HSPJ. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding HSPJ may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of HSPJ will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which HSPJ may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding HSPJ may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing HSPJ in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding HSPJ. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding HSPJ, its initiation codon, and upstream sequences are insertedinto the appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressHSPJ may be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk³¹ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C.A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding HSPJ isinserted within a marker gene sequence, transformed cells containingsequences encoding HSPJ can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding HSPJ under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding HSPJ and express HSPJ may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA--DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding HSPJ can be detectedby DNA--DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding HSPJ. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding HSPJ to detect transformantscontaining DNA or RNA encoding HSPJ.

A variety of protocols for detecting and measuring the expression ofHSPJ, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson HSPJ is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HSPJ includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding HSPJ, or anyfragments thereof may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HSPJ may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHSPJ may be designed to contain signal sequences which direct secretionof HSPJ through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding HSPJ to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and HSPJ may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingHSPJ and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying HSPJ from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of HSPJ may be producedby direct peptide synthesis using solid-phase techniques (Merrifield J.(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431 A PeptideSynthesizer (Perkin Elmer). Various fragments of ABBR may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

Therapeutics

Chemical and structural homology exits between HSPJ1 and human DNAJ-2(GI 306714). In addition, HSPJ1 is expressed in tumors and in diseasesinvolving inflammation and the immune system. Expression of HSPJ1 may beassociated with the cascade of events that initiate and maintaininflammatory and immune responses. Therefore, HSPJ1 appears to play arole in cancer and inflammatory and immune responses. The protectiveeffect of HSPJ1, however, may also be utilized to protect normal cellsand tissues from the stress caused by pathological or cellularprocesses.

Therefore, in one embodiment, antagonists which decrease the activity ofHSPJ1 may be administered to a subject to prevent or treat cancer. Suchcancers may include, but are not limited to, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma andparticularly cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus. In one aspect, antibodies which specifically bind HSPJ1 may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress HSPJ1.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding HSPJ1 may be administered to a subject to treator prevent cancer, and in particular, those cancers described above.

In another embodiment, antagonists which decrease the activity of HSPJ1may be administered to a subject to prevent or treat an immune disorder.Such a disorder may include, but is not limited to, AIDS, Addison'sdisease, adult respiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, atrophic gastritis, glomerulonephritis, gout, Graves'disease, hypereosinophilia, irritable bowel syndrome, lupuserytematosus, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding HSPJ1 may be administered to a subject to treator prevent an immune disorder, and in particular, the immune disordersdescribed above.

In one embodiment, HSPJ1 or a fragment or derivative thereof may beadministered to a subject to treat or prevent tissue damage. Tissuedamage may result from any cause, and in particular, may be associatedwith disorders which include, but are not limited to, ankylosingspondylitis, heart attacks, ischemia, damage to cells such as heartmuscle and nerve cells caused by ischemia, free radicals, toxins, andultraviolet exposure, wound healing, and insulin dependent diabetes.

In another embodiment, a vector capable of expressing HSPJ1, or afragment or a derivative thereof, may also be administered to a subjectto treat tissue damage, and in particular, the tissue damage-associateddisorders described above.

In still another embodiment, an agonist which modulates the activity ofHSPJ1 may also be administered to a subject to treat tissue damage, andin particular, the tissue damage-associated disorders described above.

Chemical and structural homology exits among HSPJ2, human HSJ1a (GI32469), and human HSJ1b (GI 32470). In addition, HSPJ2 is expressed intumors and in diseases involving the inflammatory and immune system.Expression of HSPJ2 may be associated with the cascade of events thatinitiate and maintain inflammatory and immune responses. Therefore,HSPJ2 appears to play a role in cancer and inflammatory and immuneresponse. The protective effect of HSPJ2, however, may also be utilizedto protect normal cells and tissues from the stress caused bypathological or cellular processes.

Therefore, in one embodiment, antagonists which decrease the activity ofHSPJ2 may be administered to a subject to prevent or treat cancer. Suchcancers may include, but are not limited to, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma andparticularly cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus. In one aspect, antibodies which specifically bind HSPJ2 may beused directly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress HSPJ2.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding HSPJ2 may be administered to a subject to treator prevent cancer, and in particular, the cancers described above.

In another embodiment, antagonists which decrease the activity of HSPJ2may be administered to a subject to prevent or treat an immune disorder.Such a disorder may include, but is not limited to, AIDS, Addison'sdisease, adult respiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, atrophic gastritis, glomerulonephritis, gout, Graves'disease, hypereosinophilia, irritable bowel syndrome, lupuserythematosus, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding HSPJ2 may be administered to a subject to treator prevent an immune disorder, and in particular, the immune disordersdescribed above.

In one embodiment, HSPJ2 or a fragment or derivative thereof may beadministered to a subject to treat or prevent tissue damage. Tissuedamage may result from any cause and in particular, may be associatedwith a disorder such as, but not limited to, ankylosing spondylitis,heart attacks, ischemia, damage to cells such as heart muscle and nervecells caused by ischemia, free radicals, toxins, and ultravioletexposure, wound healing, and insulin dependent diabetes.

In another embodiment, a vector capable of expressing HSPJ2, or afragment or a derivative thereof, may also be administered to a subjectto treat tissue damage, and in particular, the tissue damage-associateddisorders described above.

In still another embodiment, an agonist which modulates the activity ofHSPJ2 may also be administered to a subject to treat tissue damage, andin particular, the tissue damage-associated disorders described above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

Antagonists or inhibitors of HSPJ may be produced using methods whichare generally known in the art. In particular, purified HSPJ may be usedto produce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind HSPJ.

Antibodies to HSPJ may be generated using methods that are well known inthe art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith HSPJ or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to HSPJ have an amino acid sequence consisting of atleast five amino acids and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of HSPJ amino acids may be fused with those of another proteinsuch as keyhole limpet hemocyanin and antibody produced against thechimeric molecule.

Monoclonal antibodies to HSPJ may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1 985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983)Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. CellBiol. 62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceHSPJ-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for HSPJ mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between HSPJ and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HSPJ epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingHSPJ, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingHSPJ may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding HSPJ. Thus,complementary molecules or fragments may be used to modulate HSPJactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments, can be designed from various locations along thecoding or control regions of sequences encoding HSPJ.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encoding HSPJ.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

Genes encoding HSPJ can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes HSPJ. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5' or regulatory regions of the gene encoding HSPJ(signal sequence, promoters, enhancers, and introns). Oligonucleotidesderived from the transcription initiation site, e.g., between positions-10 and +10 from the start site, are preferred. Similarly, inhibitioncan be achieved using "triple helix" base-pairing methodology. Triplehelix pairing is useful because it causes inhibition of the ability ofthe double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Thecomplementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding HSPJ.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HSPJ. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, ex vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections orpolycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of HSPJ, antibodies toHSPJ, mimetics, agonists, antagonists, or inhibitors of HSPJ. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other is plants; cellulose,such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HSPJ, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HSPJ or fragments thereof, antibodies of HSPJ,agonists, antagonists or inhibitors of HSPJ, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind HSPJ may beused for the diagnosis of conditions or diseases characterized byexpression of HSPJ, or in assays to monitor patients being treated withHSPJ, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for HSPJ includemethods which utilize the antibody and a label to detect HSPJ in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring HSPJare known in the art and provide a basis for diagnosing altered orabnormal levels of HSPJ expression. Normal or standard values for HSPJexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHSPJ under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric means. Quantities of HSPJ expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHSPJ may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHSPJ may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of HSPJ,and to monitor regulation of HSPJ levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HSPJ or closely related molecules, may be used to identifynucleic acid sequences which encode HSPJ. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding HSPJ, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe HSPJ encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring HSPJ.

Means for producing specific hybridization probes for DNAs encoding HSPJinclude the cloning of nucleic acid sequences encoding HSPJ or HSPJderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding HSPJ may be used for the diagnosis ofconditions, disorders, or diseases which are associated with expressionof HSPJ. Examples of such conditions or diseases include cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma and particularly cancers of the adrenal gland, bladder,bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen,testis, thymus, thyroid, and uterus; inflammatory and immune disorderssuch as AIDS, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitus,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections and trauma, ankylosing spondylitis,heart attacks, ischemia, damage to cells such as heart muscle and nervecells caused by ischemia, free radicals, toxins, and ultravioletexposure, wound healing, and insulin dependent diabetes. Thepolynucleotide sequences encoding HSPJ may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dipstick, pin, ELISA assays or microarraysutilizing fluids or tissues from patient biopsies to detect altered HSPJexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding HSPJ may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingHSPJ may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding HSPJ in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of HSPJ, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes HSPJ, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HSPJ may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5'→3') and another with antisense (3'←5'),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of HSPJinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or calorimetricresponse gives rapid quantitation.

In further embodiments, oligonucleotides derived from any of thepolynucleotide sequences described herein may be used as probes inmicroarrays. The microarrays can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of i disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents.

In one embodiment, the microarray is prepared and used according to themethods described in PCT application WO95/11995 (Chee et al.), Lockhart,D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al.(1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which areincorporated herein in their entirety by reference.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs fixed to a solidsupport. Microarrays may contain oligonucleotides which cover the known5', or 3', sequence, or contain sequential oligonucleotides which coverthe full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of the sequence is knownor that are specific to one or more unidentified cDNAs which are commonto a particular cell type, developmental or disease state.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5' or more preferably at the 3' end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. The oligomers are synthesized atdesignated areas on a substrate using a light-directed chemical process.The substrate may be paper, nylon or other type of membrane, filter,chip, glass slide or any other suitable solid support.

In another aspect, the oligomers may be synthesized on the surface ofthe substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a "gridded" array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available devises (slot blot or dot blotapparatus) materials and machines (including robotic instruments) andcontain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144dots, or any other multiple which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using the microarrays, the RNA orDNA from a biological sample is made into hybridization probes. The mRNAis isolated, and cDNA is produced and used as a template to makeantisense RNA (aRNA). The aRNA is amplified in the presence offluorescent nucleotides, and labeled probes are incubated with themicroarray so that the probe sequences hybridize to complementaryoligonucleotides of the microarray. Incubation conditions are adjustedso that hybridization occurs with precise complementary matches or withvarious degrees of less complementarity. After removal of nonhybridizedprobes, a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies on the sequences, mutations, variants, or polymorphisms amongsamples.

In another embodiment of the invention, the nucleic acid sequences whichencode HSPJ may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome or to artificial chromosome constructions, such as humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial PI constructions orsingle chromosome cDNA libraries as reviewed in Price, C. M. (1993)Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

Fluorescent in situ hybridization (FISH as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, NY) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in various scientific journals or at Online Mendelian Inheritancein Man (OMIM). Correlation between the location of the gene encodingHSPJ on a physical chromosomal map and a specific disease , orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a is particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, HSPJ, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenHSPJ and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to HSPJ large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with HSPJ1 or HSPJ2, or fragments thereof, and washed. BoundHSPJ1 or HSPJ2 is then detected by methods well known in the art.Purified HSPJ1 or HSPJ2 can also be coated directly onto plates for usein the aforementioned drug screening techniques. Alternatively,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HSPJ1 or HSPJ2specifically compete with a test compound for binding HSPJ1 or HSPJ2. Inthis manner, the antibodies can be used to detect the presence of anypeptide which shares one or more antigenic determinants with HSPJ1 orHSPJ2.

In additional embodiments, the nucleotide sequences which encode HSPJ1or HSPJ2 may be used in any molecular biology techniques that have yetto be developed, provided the new techniques rely on properties ofnucleotide sequences that are currently known, including, but notlimited to, such properties as the triplet genetic code and specificbase pair interactions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I cDNA Library Construction

SYNORAB01 The cDNA library for SYNORAB01 was constructed from total RNAfrom rheumatoid synovial tissue, UC Davis (lot #48). The frozen tissuewas ground in a mortar and pestle and lysed immediately in a buffercontaining guanidinium isothiocyanate. The lysate was extracted twicewith phenol chloroform at pH 8.0 and centrifuged over a CsCl cushionusing a Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (BeckmanInstruments). The RNA was precipitated using 0.3 M sodium acetate and2.5 volumes of ethanol and resuspended in water.

The RNA was prepared with the SuperScript Plasmid System for cDNASynthesis and Plasmid Cloning (catalogue #18248-013; GIBCO/BRL,Gaithersburg Md.) with the recommended protocol. cDNAs were fractionatedon a Sepharose CL4B column (catalog #275105, Pharmacia), and those cDNAsexceeding 1 kb were ligated into PSPORT1. The plasmid was transformedinto chemically competent DH5a host cells (GIBCO/BRL, Gaithersburg,Md.).

First strand cDNA synthesis was accomplished using an oligo d(T)primer/linker which also contained an Xhol restriction site. Secondstrand synthesis was performed using a combination of DNA polymerase I,E. coli ligase and RNase H, followed by the addition of an EcoRI adaptorto the blunt ended cDNA. The EcoRI adapted, double-stranded cDNA wasthen digested with Xhol restriction enzyme and fractionated on SephacrylS400 to obtain sequences which exceeded 1000 bp in size. The sizeselected cDNAs were inserted into the LAMBDAZAP vector system(Stratagene); and the vector, which contains the PBLUESCRIPT phagemid(Stratagene), was transformed into cells of E. coli, strain XL1-BLUEMRF(Stratagene).

HNT2RAT01

The HNT2RAT01 cDNA library was prepared from the hNT2 cell line whichexhibits characteristics of a committed neuronal precursor cell which isat an early stage of development. The hNT2 cell line can be induced byretinoic acid (RA) to differentiate, as described in Andrews PW (1984)Dev Biol 103:285-293. For purposes of this invention, hNT2 cells wereinduced with RA by one of two procedures. The method used in the presentinvention involved suspending hNT2 cells in Dulbecco's modified Eagle'smedium (DMEM) including 10% fetal bovine serum and penicillin/streptomycin, treating the cells with 10 μM RA for 24 hours, andharvesting the cells immediately.

Stratagene isolated the mRNA and prepared the cDNA library. cDNAs wereprimed using oligo d(T) and size fractionated to isolate fragments of500 bp and larger. Synthetic adapter oligonucleotides were ligated ontothe cDNA molecules enabling them to be inserted into the UNI-ZAP vectorsystem (Stratagene).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was purified using the Miniprep Kit (Catalogue # 77468,Advanced Genetic Technologies Corporation, Gaithersburg Md.). Therecommended protocol included with the kit was employed except for thefollowing changes. Each of the 96 wells was filled with only 1 ml ofsterile Terrific Broth (Catalog # 22711, GIBCO/BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%. After the wells were inoculated, thebacteria were cultured for 24 hours and lysed with 60 μl of lysisbuffer. A centrifugation step (Beckman GS-6R @2900 rpm for 5 min;Beckman Instruments) was performed before the contents of the block wereadded to the primary filter plate. The optional step of addingisopropanol to TRIS buffer was not routinely performed. After the laststep in the protocol, samples were transferred to a Beckman 96-wellblock for storage.

The cDNAs were sequenced by the method of Sanger F and AR Coulson (1975;J Mol Biol 94:441 f), using a Hamilton Micro Lab 2200 (Hamilton, RenoNev.) in combination with four Peltier Thermal Cyclers (PTC200 from MJResearch, Watertown Mass.) and Applied Biosystems 377 or 373 DNASequencing Systems (Perkin Elmer) and reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences of the Sequence Listing or amino acid sequencesdeduced from them were used as query sequences against databases such asGenBank, SwissProt, BLOCKS, and Pima II. These databases which containpreviously identified and annotated sequences were searched for regionsof homology (similarity) using BLAST, which stands for Basic LocalAlignment Search Tool (Altschul, S. F. (1993) J. Mol. Evol. 36:290-300;Altschul et al. (1990) J. Mol. Biol. 215:403-410).

BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal or plant) origin. Other algorithms such asthe one described in Smith RF and TF Smith (1992; Protein Engineering5:35-51), incorporated herein by reference, can be used when dealingwith primary sequence patterns and secondary structure gap penalties. Asdisclosed in this application, the sequences have lengths of at least 49nucleotides, and no more than 12% uncalled bases (where N is recordedrather than A, C, G, O or T).

The BLAST approach, as detailed in Karlin, S. and S. F. Altschul (1993;Proc Nat. Acad. Sci. 90:5893-3) and incorporated herein by reference,searches for matches between a query sequence and a database sequence,to evaluate the statistical significance of any matches found, and toreport only those matches which satisfy the user-selected threshold ofsignificance. In this application, threshold was set at 10⁻²⁵ ornucleotides and 10⁻¹⁴ for peptides.

Incyte nucleotide sequences were searched against the GenBapdk databasesfor primate (pri), rodent (rod), and mammalian sequences (mam), anddeduced amino acid sequences from the same clones are searched againstGenBank functional protein databases, mammalian (mamp), vertebrate(vrtp) and eukaryote (eukp), for homology. The relevant database for aparticular match were reported as a Glxxx±p (where xxx is pri, rod, etcand if present, p=peptide). Product score, the calculation of which isshown below, was used to determine the electronic stringency. For anexact match, product score was set at 70 with a conservative lower limitset at approximately 40 (1-2% error due to uncalled bases).

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding HSPJ1 or HSPJ2 occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

V Extension of HSPJ Encoding Polynucleotides

The nucleic acid sequences of Incyte Clone 136466 or Incyte Clone 260873were used to design oligonucleotide primers for extending a partialnucleotide sequence to full length. One primer was synthesized toinitiate extension in the antisense direction, and the other wassynthesized to extend sequence in the sense direction. Primers were usedto facilitate the extension of the known sequence "outward" generatingamplicons containing new, unknown nucleotide sequence for the region ofinterest. The initial primers were designed from the cDNA using OLIGO4.06 (National Biosciences), or another appropriate program, to be about22 to about 30 nucleotides in length, to have a GC content of 50% ormore, and to anneal to the target sequence at temperatures of about 68°to about 72° C. Any stretch of nucleotides which would result in hairpinstructures and primer-primer dimerizations was avoided.

Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. Beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, PCR was performedusing the Peltier Thermal Cycler (PTC200; M. J. Research, Watertown,Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                                 Step 2 65° C. for 1 min                                    Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat step 4-6 for 15 additional cycles                               Step 8 94° C. for 1 sec                                                Step 9 65° C. for 1 min                                                 Step 10 68° C. for 7:15 min                                            Step 11 Repeat step 8-10 for 12 cycles                                        Step 12 72° C. for 8 min                                               Step 13 4° C. (and holding)                                         ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQUICK (QIAGEN Inc., Chatsworth, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2× Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2× Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                             Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2-4 for an additional 29 cycles                           Step 6 72° C. for 180 sec                                              Step 7 4° C. (and holding)                                           ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 and SEQ ID NO:4are used to obtain 5' regulatory sequences using the procedure above,oligonucleotides designed for 5' extension, and an appropriate genomiclibrary.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 and SEQ ID NO:4 areemployed to screen cDNAs, genomic DNAs, or mRNAs. Although the labelingof oligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³² P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A aliquot containing10⁷ counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1,or Pvu II; DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1 × salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Microarrays

To produce oligonucleotides for a microarray, the nucleotide sequencedescribed herein is examined using a computer algorithm which starts atthe 3' end of the nucleotide sequence. The algorithm identifiesoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that would interfere with hybridization. Thealgorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J.D. et al., PCT/WO95/25 116, incorporated herein byreference). In another alternative, a "gridded" array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the micro-array.

VIII Complementary Polynucleotides

Sequence complementary to the HSPJ1 or HSPJ2-encoding sequence, or anypart thereof, is used to decrease or inhibit expression of naturallyoccurring HSPJ1 or HSPJ2. Although use of oligonucleotides comprisingfrom about 15 to about 30 base-pairs is described, essentially the sameprocedure is used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using Oligo 4.06 software and the codingsequence of HSPJ1 or HSPJ2, SEQ ID NO:1 or SEQ ID NO:3. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5' sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the HSPJ1 or HSPJ2-encodingtranscript.

IX Expression of HSPJ

Expression of HSPJ1 or HSPJ2 is accomplished by subcloning the cDNAsinto appropriate vectors and transforming the vectors into host cells.In this case, the cloning vector is also used to express HSPJ1 or HSPJ2in E. coli. Upstream of the cloning site, this vector contains apromoter for B3-galactosidase, followed by sequence containing theamino-terminal Met, and the subsequent seven residues ofB3-galactosidase. Immediately following these eight residues is abacteriophage promoter useful for transcription and a linker containinga number of unique restriction sites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofHSPJ1 or HSPJ2 into the bacterial growth media which can be useddirectly in the following assay for activity.

X Demonstration of HSPJ Activity

HSPJ1 or HSPJ2 induction by heat or toxins may be a demonstrated usingprimary cultures of human fibroblasts or human cell lines such asCCL-13, HEK293, or HEP G2 (ATCC). To heat induce HSPJ1 or HSPJ2expression, aliquots of cells are incubated at 42° C. for 15, 30, or 60minutes, control aliquots are incubated at 37° C. for the same timeperiods. To induce HSPJ1 or HSPJ2 expression by toxins, aliquots ofcells are treated with 100 μM arsenite or 20 mM azetidine-2-carboxylicacid for 0, 3, 6, or 12 hours. After exposure to heat, arsinate, or theamino acid analogue, samples of the treated cells are harvested and celllysates prepared for analysis by Western blot.

Cells are lysed in lysis buffer containing 1% Nonidet P-40, 0.15 M NaCl,50 mM Tris-HCl, 5 mM EDTA, 2 mMN-ethylmaleimide, 2 mMphenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 1 mg/ml pepstatin.Twenty micrograms of the cell lysate is separated on an 8% SDS-PAGE geland transferred to a nitrocellulose membrane. After blocking with 5%nonfat dry milk/phosphate-buffered saline for 1 h, the membrane isincubated overnight at 4° C. or at room temperature for 2-4 hours with a1:1000 dilution of anti-HSPJ1 or HSPJ2 serum in 2% nonfat drymilk/phosphate-buffered saline. The membrane is then washed and loincubated with a 1:1000 dilution of horseradish peroxidase-conjugatedgoat anti-rabbit IgG (Cappel) in 2% dry milk/phosphate-buffered saline.After washing with 0. 1% Tween 20 in phosphate-buffered saline, theHSPJ1 or HSPJ2 protein is detected and compared to controls by using theECL system (Amersham).

XI Production of HSPJ Specific Antibodies

HSPJ1 or HSPJ2 that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 or SEQ ID NO:4 is analyzed usingDNASTAR software (DNASTAR Inc) to determine regions of highimmunogenicity and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art.Selection of appropriate epitopes, such as those near the C-terminus orin hydrophilic regions, is described by Ausubel et al. (supra), andothers.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 43 1A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio iodinated, goat anti-rabbitIgG.

XII Purification of Naturally Occurring HSPJ Using Specific Antibodies

Naturally occurring or recombinant HSPJ1 or HSPJ2 is substantiallypurified by immunoaffinity chromatography using antibodies specific forHSPJ1 or HSPJ2. An immunoaffinity column is constructed by covalentlycoupling HSPJ1 or HSPJ2 antibody to an activated chromatographic resin,such as CNBr-activated Sepharose (Pharmacia & Upjohn). After thecoupling, the resin is blocked and washed according to themanufacturer's instructions.

Media containing HSPJ1 or HSPJ2 is passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of HSPJ 1 or HSPJ2 (e.g., high ionic strengthbuffers in the presence of detergent). The column is eluted underconditions that disrupt antibody/HSPJ1 or HSPJ2 binding (e.g., a bufferof pH 2-3 or a high concentration of a chaotrope, such as urea orthiocyanate ion), and HSPJ 1 or HSPJ2 is collected.

XIII Identification of Molecules Which Interact with HSPJ

HSPJ1 or HSPJ2 or biologically active fragments thereof are labeled with¹²⁵ I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled HSPJ1 or HSPJ2, washed and anywells with labeled HSPJ1 or HSPJ2 complex are assayed. Data obtainedusing different concentrations of HSPJ1 or HSPJ2 are used to calculatevalues for the number, affinity, and association of HSPJ1 or HSPJ2 withthe candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 7                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 358 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: SYN0RAB01                                                        (B) CLONE: 136466                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Ala Pro Gln Asn Leu Ser Thr Phe Cys Le - #u Leu Leu Leu Tyr        Leu                                                                              1               5  - #                10  - #                15              - - Ile Gly Ala Val Ile Ala Gly Arg Asp Phe Ty - #r Lys Ile Leu Gly Val                  20      - #            25      - #            30                   - - Pro Arg Ser Ala Ser Ile Lys Asp Ile Lys Ly - #s Ala Tyr Arg Lys Leu              35          - #        40          - #        45                       - - Ala Leu Gln Leu His Pro Asp Arg Asn Pro As - #p Asp Pro Gln Ala Gln          50              - #    55              - #    60                           - - Glu Lys Phe Gln Asp Leu Gly Ala Ala Tyr Gl - #u Val Leu Ser Asp Ser      65                  - #70                  - #75                  - #80        - - Glu Lys Arg Lys Gln Tyr Asp Thr Tyr Gly Gl - #u Glu Gly Leu Lys Asp                      85  - #                90  - #                95               - - Gly His Gln Ser Ser His Gly Asp Ile Phe Se - #r His Phe Phe Gly Asp                  100      - #           105      - #           110                  - - Phe Gly Phe Met Phe Gly Gly Thr Pro Arg Gl - #n Gln Asp Arg Asn Ile              115          - #       120          - #       125                      - - Pro Arg Gly Ser Asp Ile Ile Val Asp Leu Gl - #u Val Thr Leu Glu Glu          130              - #   135              - #   140                          - - Val Tyr Ala Gly Asn Phe Val Glu Val Val Ar - #g Asn Lys Pro Val Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Gln Ala Pro Gly Lys Arg Lys Cys Asn Cy - #s Arg Gln Glu Met        Arg                                                                                             165  - #               170  - #               175             - - Thr Thr Gln Leu Gly Pro Gly Arg Phe Gln Me - #t Thr Gln Glu Val Val                  180      - #           185      - #           190                  - - Cys Asp Glu Cys Pro Asn Val Lys Leu Val As - #n Glu Glu Arg Thr Leu              195          - #       200          - #       205                      - - Glu Val Glu Ile Glu Pro Gly Val Arg Asp Gl - #y Met Glu Tyr Pro Phe          210              - #   215              - #   220                          - - Ile Gly Glu Gly Glu Pro His Val Asp Gly Gl - #u Pro Gly Asp Leu Arg      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Phe Arg Ile Lys Val Val Lys His Pro Ile Ph - #e Glu Arg Arg Gly        Asp                                                                                             245  - #               250  - #               255             - - Asp Leu Tyr Thr Asn Val Thr Val Ser Leu Va - #l Glu Ser Leu Val Gly                  260      - #           265      - #           270                  - - Phe Glu Met Asp Ile Thr His Leu Asp Gly Hi - #s Lys Val His Ile Ser              275          - #       280          - #       285                      - - Arg Asp Lys Ile Thr Arg Pro Gly Ala Xaa Xa - #a Trp Lys Lys Gly Glu          290              - #   295              - #   300                          - - Gly Leu Pro Asn Phe Asp Asn Asn Asn Ile Ly - #s Gly Ser Leu Ile Ile      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Thr Phe Asp Val Asp Phe Pro Lys Glu Gln Le - #u Thr Glu Glu Ala        Arg                                                                                             325  - #               330  - #               335             - - Glu Gly Ile Lys Gln Leu Leu Lys Gln Gly Se - #r Val Gln Lys Val Tyr                  340      - #           345      - #           350                  - - Asn Gly Leu Gln Gly Tyr                                                          355                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1376 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: SYN0RAB01                                                        (B) CLONE: 136466                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TCTCACCGGG ACTCGGGACT CCCGGGAAGT GGACCGGCAG AAGAGGGGGC TA -             #GCTAGCTG     60                                                                 - - TCTCTGCGGA CCAGGGAGAC CCCCGCGCCC CCCCGGTGTG AGGCGGCCTC AC -            #AGGGCCGG    120                                                                 - - GTGGGCTGGC GAGCCGACGC GGCGGCGGAG GAGGCTGTGA GGAGTGTGTG GA -            #ACAGGACC    180                                                                 - - CGGGACAGAG GAACCATGGC TCCGCAGAAC CTGAGCACCT TTTGCCTGTT GC -            #TGCTATAC    240                                                                 - - CTCATCGGGG CGGTGATTGC CGGACGAGAT TTCTATAAGA TCTTGGGGGT GC -            #CTCGAAGT    300                                                                 - - GCCTCTATAA AGGATATTAA AAAGGCCTAT AGGAAACTAG CCCTGCAGCT TC -            #ATCCCGAC    360                                                                 - - CGGAACCCTG ATGATCCACA AGCCCAGGAG AAATTCCAGG ATCTGGGTGC TG -            #CTTATGAG    420                                                                 - - GTTCTGTCAG ATAGTGAGAA ACGGAAACAG TACGATACTT ATGGTGAAGA AG -            #GATTAAAA    480                                                                 - - GATGGTCATC AGAGCTCCCA TGGAGACATT TTTTCACACT TCTTTGGGGA TT -            #TTGGTTTC    540                                                                 - - ATGTTTGGAG GAACCCCTCG TCAGCAAGAC AGAAATATTC CAAGAGGAAG TG -            #ATATTATT    600                                                                 - - GTAGATCTAG AAGTCACTTT GGAAGAAGTA TATGCAGGAA ATTTTGTGGA AG -            #TAGTTAGA    660                                                                 - - AACAAACCTG TGGCAAGGCA GGCTCCTGGC AAACGGAAGT GCAATTGTCG GC -            #AAGAGATG    720                                                                 - - CGGACCACCC AGCTGGGCCC TGGGCGCTTC CAAATGACCC AGGAGGTGGT CT -            #GCGACGAA    780                                                                 - - TGCCCTAATG TCAAACTAGT GAATGAAGAA CGAACGCTGG AAGTAGAAAT AG -            #AGCCTGGG    840                                                                 - - GTGAGAGACG GCATGGAGTA CCCCTTTATT GGAGAAGGTG AGCCTCACGT GG -            #ATGGGGAG    900                                                                 - - CCTGGAGATT TACGGTTCCG AATCAAAGTT GTCAAGCACC CAATATTTGA AA -            #GGAGAGGA    960                                                                 - - GATGATTTGT ACACAAATGT GACAGTCTCA TTAGTTGAGT CACTGGTTGG CT -            #TTGAGATG   1020                                                                 - - GATATTACTC ACTTGGATGG TCACAAGGTA CATATTTCCC GGGATAAGAT CA -            #CCAGGCCA   1080                                                                 - - GGAGCGAANT ANTGGAAGAA AGGGGAAGGG CTCCCCAACT TTGACAACAA CA -            #ATATCAAG   1140                                                                 - - GGCTCTTTGA TAATCACTTT TGATGTGGAT TTTCCAAAAG AACAGTTAAC AG -            #AGGAAGCG   1200                                                                 - - AGAGAAGGTA TCAAACAGCT ACTGAAACAA GGGTCAGTGC AGAAGGTATA CA -            #ATGGACTG   1260                                                                 - - CAAGGATATT GAGAGTGAAT AAAATTGGAC TTTGTTTAAA ATAAGTGAAT AA -            #GCGATATT   1320                                                                 - - TATTATCTGC AAGGTTTTTT TGTGTGTGTT TTTGTTTTTA TTTTCAATAT GC - #AAGT           1376                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 330 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: HNT2RAT01                                                        (B) CLONE: 260873                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Met Val Asp Tyr Tyr Glu Val Leu Gly Val Gl - #n Arg His Ala Ser Pro       1               5  - #                10  - #                15               - - Glu Asp Ile Lys Lys Ala Tyr Arg Lys Leu Al - #a Leu Lys Trp His Pro                  20      - #            25      - #            30                   - - Asp Lys Asn Pro Glu Asn Lys Glu Glu Ala Gl - #u Arg Lys Phe Lys Gln              35          - #        40          - #        45                       - - Val Ala Glu Ala Tyr Glu Val Leu Ser Asp Al - #a Lys Lys Arg Asp Ile          50              - #    55              - #    60                           - - Tyr Asp Lys Tyr Gly Lys Glu Gly Leu Asn Gl - #y Gly Gly Gly Gly Gly      65                  - #70                  - #75                  - #80        - - Ser His Phe Asp Ser Pro Phe Glu Phe Gly Ph - #e Thr Phe Arg Asn Pro                      85  - #                90  - #                95               - - Asp Asp Val Phe Arg Glu Phe Phe Gly Gly Ar - #g Asp Pro Phe Ser Phe                  100      - #           105      - #           110                  - - Asp Phe Phe Glu Asp Pro Phe Glu Asp Phe Ph - #e Gly Asn Arg Arg Gly              115          - #       120          - #       125                      - - Pro Arg Gly Ser Arg Ser Arg Gly Thr Gly Se - #r Phe Phe Ser Ala Phe          130              - #   135              - #   140                          - - Ser Gly Phe Pro Ser Phe Gly Ser Gly Phe Se - #r Ser Phe Asp Thr Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Phe Thr Ser Phe Gly Ser Leu Gly His Gly Gl - #y Leu Thr Ser Phe        Ser                                                                                             165  - #               170  - #               175             - - Ser Thr Ser Phe Gly Gly Ser Gly Met Gly As - #n Phe Lys Ser Ile Ser                  180      - #           185      - #           190                  - - Thr Ser Thr Lys Met Val Asn Gly Arg Lys Il - #e Thr Thr Lys Arg Ile              195          - #       200          - #       205                      - - Val Glu Asn Gly Gln Glu Arg Val Glu Val Gl - #u Glu Asp Gly Gln Leu          210              - #   215              - #   220                          - - Lys Ser Leu Thr Ile Asn Gly Val Ala Asp As - #p Asp Ala Leu Xaa Glu      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Glu Arg Met Arg Arg Gly Gln Asn Val Leu Pr - #o Ala Gln Pro Ala        Gly                                                                                             245  - #               250  - #               255             - - Leu Arg Pro Pro Lys Pro Pro Arg Pro Ala Se - #r Leu Leu Arg His Xaa                  260      - #           265      - #           270                  - - Pro His Cys Leu Ser Lys Glu Glu Gly Glu Gl - #n Asp Arg Pro Trp Ala              275          - #       280          - #       285                      - - Pro Xaa Xaa Trp Xaa Pro Leu Ala Ser Xaa Al - #a Gly Xaa Xaa Glu Gly          290              - #   295              - #   300                          - - Xaa Lys Arg Met Xaa Ala Glu Ala Glu Arg Gl - #y Val Glu Glu Glu Glu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Val Asp Gln Arg Gln Ser Leu Asp Arg Thr                                                  325  - #               330                                     - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1330 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: HNT2RAT01                                                        (B) CLONE: 260873                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CGNAGGAGAG NAAAGGAAAG NCGCCGCAGG AGCCGCCGCN ACCACCAGCG NC -            #ACANTCCT     60                                                                 - - GGNGCTNTGA GGAGATTCGG GCCGTCACCC TGCCTCCCCT GCTTCCCGCC AC -            #CGGCCGCT    120                                                                 - - TCTTTCCTCG GACCCATTCC AACAATCTCG TAAAACATGG TGGATTACTA TG -            #AAGTTCTA    180                                                                 - - GGCGTGCAGA GACATGCCTC ACCCGAGGAT ATTAAAAAGG CATATCGGAA AC -            #TGGCACTG    240                                                                 - - AAGTGGCATC CAGATAAAAA TCCTGAGAAT AAAGAAGAAG CAGAGAGAAA AT -            #TCAAGCAA    300                                                                 - - GTAGCGGAGG CATATGAAGT GCTGTCGGAT GCTAAGAAAC GGGACATCTA TG -            #ACAAATAT    360                                                                 - - GGCAAAGAAG GATTAAATGG TGGNGGNGGN GGTGGAAGTC ATTTTGACAG TC -            #CATTTGAA    420                                                                 - - TTTGGCTTCA CATTCCGTAA CCCAGATGAT GTCTTCAGGG AATTTTTTGG TG -            #GAAGGGAC    480                                                                 - - CCATTTTCAT TTGACTTCTT TGAAGACCCT TTTGAGGACT TCTTTGGGAA TC -            #GAAGGGGT    540                                                                 - - CCCCGAGGAA GCAGAAGCCG AGGGACGGGG TCGTTTTTCT CTGCGTTCAG TG -            #GATTTCCG    600                                                                 - - TCTTTTGGAA GTGGATTTTC TTCTTTTGAT ACAGGATTTA CTTCATTTGG GT -            #CACTAGGT    660                                                                 - - CACGGGGGCC TCACTTCATT CTCTTCCACG TCATTTGGTG GTAGTGGCAT GG -            #GCAACTTC    720                                                                 - - AAATCGATAT CAACTTCAAC TAAAATGGTT AATGGCAGAA AAATCACTAC AA -            #AGAGAATT    780                                                                 - - GTCGAGAACG GTCAAGAAAG AGTAGAAGTT GAAGAAGATG GCCAGTTAAA GT -            #CCTTAACA    840                                                                 - - ATAAATGGTG TKGCCGACGA CGATGCCCTC GSTGAGGAGC GCATGCGGAG AG -            #GCCAGAAC    900                                                                 - - GTCCTGCCAG CCCAGCCTGC CGGCCTCCGA CCGCCGAAGC CGCCCCGGCC TG -            #CCTCGTTG    960                                                                 - - CTGAGACACG NGCCTCATTG TCTCTCTAAG GAGGAGGGCG AGCAGGACCG AC -            #CTTGGGCA   1020                                                                 - - CCCGNGNCCT GGNNCCCCCT CGCTTCCNCA GCAGGNTTNN AAGAAGGTNG CA -            #AGAGGATG   1080                                                                 - - NAAGCAGAAG CAGAGAGAGG AGTCGAAGAA GAAGAAGTCG ACCAAAGGCA AT -            #CACTAGAC   1140                                                                 - - CGGACTTGAG GCACGCGGTG CACCCCCAGA CGCTGGCGCT CCACCGTGCT CG -            #GCATGCGG   1200                                                                 - - TCGTGCACAC GCGCTAGGTA GCAGCGTCGG TCAGGACTGT CTCGAGGCCA CA -            #CTCGCTCG   1260                                                                 - - GCAGGATTAT GCGATCACGG ATCAGTCAGA GCAGGGTCAG GAGACGGGGC TG -            #ACGGCACG   1320                                                                 - - GGTGGCGGGG                - #                  - #                      - #      1330                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 397 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 306714                                                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Met Val Lys Glu Thr Thr Tyr Tyr Asp Val Le - #u Gly Val Lys Pro Asn       1               5  - #                10  - #                15               - - Ala Thr Gln Glu Glu Leu Lys Lys Ala Tyr Ar - #g Lys Leu Ala Leu Lys                  20      - #            25      - #            30                   - - Tyr His Pro Asp Lys Asn Pro Asn Glu Gly Gl - #u Lys Phe Lys Gln Ile              35          - #        40          - #        45                       - - Ser Gln Ala Tyr Glu Val Leu Ser Asp Ala Ly - #s Lys Arg Glu Leu Tyr          50              - #    55              - #    60                           - - Asp Lys Gly Gly Glu Gln Ala Ile Lys Glu Gl - #y Gly Ala Gly Gly Gly      65                  - #70                  - #75                  - #80        - - Phe Gly Ser Pro Met Asp Ile Phe Asp Met Ph - #e Phe Gly Gly Gly Gly                      85  - #                90  - #                95               - - Arg Met Gln Arg Glu Arg Arg Gly Lys Asn Va - #l Val His Gln Leu Ser                  100      - #           105      - #           110                  - - Val Thr Leu Glu Asp Leu Tyr Asn Gly Ala Th - #r Arg Lys Leu Ala Leu              115          - #       120          - #       125                      - - Gln Lys Asn Val Ile Cys Asp Lys Cys Glu Gl - #y Arg Gly Gly Lys Lys          130              - #   135              - #   140                          - - Gly Ala Val Glu Cys Cys Pro Asn Cys Arg Gl - #y Thr Gly Met Gln Ile      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Ile His Gln Ile Gly Pro Gly Met Val Gl - #n Gln Ile Gln Ser        Val                                                                                             165  - #               170  - #               175             - - Cys Met Glu Cys Gln Gly His Gly Glu Arg Il - #e Ser Pro Lys Asp Arg                  180      - #           185      - #           190                  - - Cys Lys Ser Cys Asn Gly Arg Lys Ile Val Ar - #g Glu Lys Lys Ile Leu              195          - #       200          - #       205                      - - Glu Val His Ile Asp Lys Gly Met Lys Asp Gl - #y Gln Lys Ile Thr Phe          210              - #   215              - #   220                          - - His Gly Glu Gly Asp Gln Glu Pro Gly Leu Gl - #u Pro Gly Asp Ile Ile      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Val Leu Asp Gln Lys Asp His Ala Val Ph - #e Thr Arg Arg Gly        Glu                                                                                             245  - #               250  - #               255             - - Asp Leu Phe Met Cys Met Asp Ile Gln Leu Va - #l Glu Ala Leu Cys Gly                  260      - #           265      - #           270                  - - Phe Gln Lys Pro Ile Ser Thr Leu Asp Asn Ar - #g Thr Ile Val Ile Thr              275          - #       280          - #       285                      - - Ser His Pro Gly Gln Ile Val Lys His Gly As - #p Ile Lys Cys Val Leu          290              - #   295              - #   300                          - - Asn Glu Gly Met Pro Ile Tyr Arg Arg Pro Ty - #r Glu Lys Gly Arg Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ile Ile Glu Phe Lys Val Asn Phe Pro Glu As - #n Gly Phe Leu Ser        Pro                                                                                             325  - #               330  - #               335             - - Asp Lys Leu Ser Leu Leu Glu Lys Leu Leu Pr - #o Glu Arg Lys Glu Val                  340      - #           345      - #           350                  - - Glu Glu Thr Asp Glu Met Asp Gln Val Glu Le - #u Val Asp Phe Asp Pro              355          - #       360          - #       365                      - - Asn Gln Glu Arg Arg Arg His Tyr Asn Gly Gl - #u Ala Tyr Glu Asp Asp          370              - #   375              - #   380                          - - Glu His His Pro Arg Gly Gly Val Gln Cys Gl - #n Thr Ser                  385                 3 - #90                 3 - #95                            - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 351 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 32469                                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Ala Ser Tyr Tyr Glu Ile Leu Asp Val Pr - #o Arg Ser Ala Ser Ala       1               5  - #                10  - #                15               - - Asp Asp Ile Lys Lys Ala Tyr Arg Arg Lys Al - #a Leu Gln Trp His Pro                  20      - #            25      - #            30                   - - Asp Lys Asn Pro Asp Asn Lys Glu Phe Ala Gl - #u Lys Lys Phe Lys Glu              35          - #        40          - #        45                       - - Val Ala Glu Ala Tyr Glu Val Leu Ser Asp Ly - #s His Lys Arg Glu Ile          50              - #    55              - #    60                           - - Tyr Asp Arg Tyr Gly Arg Glu Gly Leu Thr Gl - #y Thr Gly Thr Gly Pro      65                  - #70                  - #75                  - #80        - - Ser Arg Ala Glu Ala Gly Ser Gly Gly Pro Gl - #y Phe Thr Phe Thr Phe                      85  - #                90  - #                95               - - Arg Ser Pro Glu Glu Val Phe Arg Glu Phe Ph - #e Gly Ser Gly Asp Pro                  100      - #           105      - #           110                  - - Phe Ala Glu Leu Phe Asp Asp Leu Gly Pro Ph - #e Ser Glu Leu Gln Asn              115          - #       120          - #       125                      - - Arg Gly Ser Arg His Ser Gly Pro Phe Phe Th - #r Phe Ser Ser Ser Phe          130              - #   135              - #   140                          - - Pro Gly His Ser Asp Phe Ser Ser Ser Ser Ph - #e Ser Phe Ser Pro Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Gly Ala Phe Arg Ser Val Ser Thr Ser Th - #r Thr Phe Val Gln        Gly                                                                                             165  - #               170  - #               175             - - Arg Arg Ile Thr Thr Arg Arg Ile Met Glu As - #n Gly Gln Glu Arg Val                  180      - #           185      - #           190                  - - Glu Val Glu Glu Asp Gly Gln Leu Lys Ser Va - #l Thr Ile Asn Gly Val              195          - #       200          - #       205                      - - Pro Asp Asp Leu Ala Arg Gly Leu Glu Leu Se - #r Arg Arg Glu Gln Gln          210              - #   215              - #   220                          - - Pro Ser Val Thr Ser Arg Ser Gly Gly Thr Gl - #n Val Gln Gln Thr Pro      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Ser Cys Pro Leu Asp Ser Asp Leu Ser Gl - #u Asp Glu Asp Leu        Gln                                                                                             245  - #               250  - #               255             - - Leu Ala Met Ala Tyr Ser Leu Ser Glu Met Gl - #u Ala Ala Gly Lys Lys                  260      - #           265      - #           270                  - - Pro Ala Gly Gly Arg Glu Ala Gln His Arg Ar - #g Gln Gly Arg Pro Arg              275          - #       280          - #       285                      - - Pro Ser Thr Lys Ile Gln Ala Trp Gly Gly Pr - #o Arg Arg Val Arg Gly          290              - #   295              - #   300                          - - Val Lys Gln Pro Asn Ala Val His Pro Gln Ar - #g Arg Arg Pro Leu Ala      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ala Ser Ser Ser Glu His Arg Ala Gln Pro As - #p Leu Ile Gln Ile        Leu                                                                                             325  - #               330  - #               335             - - Thr Gly Gly Ser Asp Ser Leu Trp Glu Glu Ly - #s Arg Gly Val Ser                      340      - #           345      - #           350                  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 277 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 32470                                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Met Ala Ser Tyr Tyr Glu Ile Leu Asp Val Pr - #o Arg Ser Ala Ser Ala       1               5  - #                10  - #                15               - - Asp Asp Ile Lys Lys Ala Tyr Arg Arg Lys Al - #a Leu Gln Trp His Pro                  20      - #            25      - #            30                   - - Asp Lys Asn Pro Asp Asn Lys Glu Phe Ala Gl - #u Lys Lys Phe Lys Glu              35          - #        40          - #        45                       - - Val Ala Glu Ala Tyr Glu Val Leu Ser Asp Ly - #s His Lys Arg Glu Ile          50              - #    55              - #    60                           - - Tyr Asp Arg Tyr Gly Arg Glu Gly Leu Thr Gl - #y Thr Gly Thr Gly Pro      65                  - #70                  - #75                  - #80        - - Ser Arg Ala Glu Ala Gly Ser Gly Gly Pro Gl - #y Phe Thr Phe Thr Phe                      85  - #                90  - #                95               - - Arg Ser Pro Glu Glu Val Phe Arg Glu Phe Ph - #e Gly Ser Gly Asp Pro                  100      - #           105      - #           110                  - - Phe Ala Glu Leu Phe Asp Asp Leu Gly Pro Ph - #e Ser Glu Leu Gln Asn              115          - #       120          - #       125                      - - Arg Gly Ser Arg His Ser Gly Pro Phe Phe Th - #r Phe Ser Ser Ser Phe          130              - #   135              - #   140                          - - Pro Gly His Ser Asp Phe Ser Ser Ser Ser Ph - #e Ser Phe Ser Pro Gly      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Ala Gly Ala Phe Arg Ser Val Ser Thr Ser Th - #r Thr Phe Val Gln        Gly                                                                                             165  - #               170  - #               175             - - Arg Arg Ile Thr Thr Arg Arg Ile Met Glu As - #n Gly Gln Glu Arg Val                  180      - #           185      - #           190                  - - Glu Val Glu Glu Asp Gly Gln Leu Lys Ser Va - #l Thr Ile Asn Gly Val              195          - #       200          - #       205                      - - Pro Asp Asp Leu Ala Arg Gly Leu Glu Leu Se - #r Arg Arg Glu Gln Gln          210              - #   215              - #   220                          - - Pro Ser Val Thr Ser Arg Ser Gly Gly Thr Gl - #n Val Gln Gln Thr Pro      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Ser Cys Pro Leu Asp Ser Asp Leu Ser Gl - #u Asp Glu Asp Leu        Gln                                                                                             245  - #               250  - #               255             - - Leu Ala Met Ala Tyr Ser Leu Ser Glu Met Gl - #u Ala Ala Gly Lys Lys                  260      - #           265      - #           270                  - - Pro Ala Asp Val Phe                                                              275                                                                  __________________________________________________________________________

What is claimed is:
 1. A substantially purified human DnaJ-like-proteinhaving a sequence selected from the group consisting of SEQ ID NO:1 andSEQ ID NO:3.
 2. A pharmaceutical composition comprising a substantiallypurified human DnaJ-like protein of claim 1 in conjunction with asuitable pharmaceutical carrier.